Production of 4n-demethyl-4n-ethyl tetracyclines



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3,364,123 Patented Jan. 16, 1968 1 Claim. (Cl. 195-80) ABSTRACT OF THEDISCLOSURE The following disclosure relates to the preparation of4N-demethyl-4N-ethyl-tetracycline and 4N-deznethyl-4N- ethyl (halo)tetracyclines which comprises culturing a 7-chlorotetracycline producingmicroorganism of the strain S. aureofaczens in the presence of excesscyanocobalamin, methionine, methionine sulfoxide and threonine.

This application is a division of application, Ser. No. 278,400, filedMay 6, 1963.

This invention relates to the production of 4N-dernethyl-4N-ethyltetracyclines. More particularly, the invention relates to the newantibiotics 4N-demethyl-4N-ethyltetracycline,4N-dernethyl-4N-ethyl-7-(halo)tetracycline, for example,4Ndemethyl-4N-ethylchlortetracycline and 4N- demethyl 4Nethylbromtetracycline, 4N-demethyl-4N- ethyl-6-demethyltetracycline, and4Ndemethyl-4N-ethyl- 6-dernethyl-7-(halojltetracycline, for example4N-demethyl-4N-ethyl-G-dimethylchlortetracycline, and4N-demethyl-4N-ethyl-6-demethylbromtetracycline, and to methods ofproducing these compounds by microbiological means.

The compound of this invention are produced by culturing strains of themicroorganism Streptomyces aureofaciens in a fermentation medium underaerobic conditions.

The conditions of fermentation are generally the same as theconventional methods of producing tetracyclines by fermentation. Thefermentation medium contains the usual nutrients and mineral sourcessupplying carbon, nitrogen and energy to the developing culture.Suitable nutrients include, for example, carbohydrates such as starch,dextrose, sucrose, glucose, molasses, and nitrogen sources such assoybean meal, yeast, meat extracts, cornsteep liquor, distillerssolubles, inorganic salts such as calcium carbonate, ammonium sulfate asWell as trace elements and other conventional subtances.

The inoculum employed for producing the4N-demethyl-4N-ethyl(halo)tetracyclines in the practice of thisinvention is prepared by suspending a sterile lyophobized pellet of themicroorganism being employed in a sterile medium of the followingcomposition (A) in flasks or bottles:

Gm./liter Soybean meal (extraction process) 30 Calcium carbonate(powdered) 7 Alkali metal halide salt 1 Glucose O Distilled water tomake 1 liter.

Growth of the inoculum in 50 ml. of medium A in a 250 ml. flask iscarried out at about C. on a rotary shaker (280 rpm, 2" strokes) forabout 72 hours. The above medium may also be employed for fermentation.

The alkali metal halide salts employed in the above medium will dependon the final product sought to be produced. Thus, where4N-dernethyl-4N-ethylchlortetracycline is the final product, salts suchas sodium chloride may be used; while4N-demethyl-4N-ethylbromtetracycline production requires the use of suchsalts as sodium bromide. It has also been found that when the alkalimetal halide salt is omitted entirely from the medium and an equivalentamount of deionized Water is substituted for the distilled Water,4N-demethyl-4N-ethyltetracycline is obtained, employing the abovemedium.

In addition to the ingredients of the foregoing fermentation medium, itis essential, in the production of the4N-demethyl-4N-ethyl(halo)tetracyclines and the4N-dernethyl-4N-ethyltetracyclines, to add small amounts of ethionine toand further amounts of a compound selected from the group consisting ofcyanocobalamin, methionine, methionine sulfoxide and threonine. Theamount of ethionine, which may be DL-, D- or L-ethionine, employed inthe practice of this invention may vary from 50 to 1000 mg. per literand is preferably about to 200 mg./liter. The amounts of the othercompounds Which are employed in combination with the ethionine may varyfrom about 200 to about 1000 mg./ liter and are preferably about 500 toabout 1500 mg./liter for methionine, methionine sulfoxide and threonine;and for cyanocobalamine from about 10 to 50 mg./liter and preferablyabout 20 to 40 mg./ liter.

The microorganisms employed in producing the 4N-demethyl-4N-ethyl(halo)tetracycline and the 4N-demethyl-4ll-ethyltetracyciine products of this invention are Szrepromyces(ZIU'EG-fllCiCilS ATCC 13900, S. aurcofaciens ATCC 13899; S.aureofncz'ens ATCC 12416a; S. aureo facz'cils ATCC 124161); S.aureofaciens ATCC l2416c; S. auricofaciens NRRL B. 1288; S. aureofaciensNRRL 2209; S. ZllIC0fflCi/"l.5' NRRL B 1286; S. wm-eofaciens NRRL B1287; and S. viridofaciens ATCC 11989.

In addition to the foregoing, it has been found that the 4N-demethyl-4ll-ethyl-6-demethyltctracyclines and the 4Ndemethyl-4N-ethyl-G-demethyl(halo) tetracyclines, for example 4l\ -demethyl-4Nethyl-6demethylchlortetracy- Cline and4N-dernethyl-4N-ethyl-6-dernethylbromtetracyclines may also be produced.The inoculum employed for biosynthesis these compounds is prepared bysuspending a sterile lyophilized pellet of the microorganism employed ina sterile medium of the following composition (B) in flasks or bottles:

Sucrose gm./liter 30 EI4)2SO4 dO 2 dO 7 Cornsteep liquor ml./liter 36.5

Distilled water to make 1 liter.

Growth of the inoculum in 50 ml. of medium B in a 250 ml. flask iscarried out at about 25 C. on a rotary shaker (280 r.p.m.; 2 stroke) forabout 72 hours.-

For fermentation, the inoculum is added to a sterile fermentation mediumof the following composition (C):

Distilled water to make 1 liter.

The fermentation is carried out at about 25 C. for 6 to 7 days, anadequate air supply being continually present.

In order to produce the 4N-demethyl-4N-ethyl-6-demethyltetracycline, theabove medium may be employed excepting that the amount of chloride ionspresent in the medium must be minimized. Thus, if an equivalent amountof soybean meal (extraction process) were substituted for the corn steepliquor and (NHQ SQ, were substituted for the NH Ci; and CO(NO weresubstituted for COCl and deionized water were substituted for thedistilled water, 4N-demethyl-4N-ethyl-6-demethyltetracycline isproduced. In addition, if 1 gm./liter of NaBr were added to the mediumemployed in the production of4N-demethyl-4N-ethyl-6-demethyltetracycline, there is obtained4N-demethyl-4N-ethyl-6-demethylbromtetracycline.

In addition to the components of the above identified fermentationmedium (C) a small but effective amount of ethionine must be present.The amount of ethionine which may be D-, L-, or DL-ethionine, which maybe used in the practice of this invention may vary from about 50 to 1000mg. per liter and is preferably from 100 to 500 mg. per liter.

The microorganisms employable to obtain the 4N-de- 9 methyl 4N ethyl 6demethyltetracyclines and 4N- demethyl 4Nethyl-6-demethyl(halo)tetracyclines such as 4N demethyl 4N ethyl 6demethylchlortetracycline and 4N demethyl 4N ethyl 6demethylbromtetracycline are S. aureofaciens ATCC 12551, S. aureofaciensATCC 12552, S. auerofaciens, ATCC 12553, and S. aureofaciens ATCC 12554.

The 4N demethyl 4N ethyl tetracyclines may be separated from the brothby extracting with organic solvents such as ethyl acetate, n-butanol,methyl isobutylketone and the like, then concentrating the rich extractto dryness by evaporization or lyophilizationJThe products may'be'further purified by cellulose column chromatography.

The presence of these novel 4N demethyl 4N ethyl i tetracyclines isdemonstrated and distinguished from other tetracyclines by paperchromatography employing the methods of Bohonos et al., AntibioticsAnnual 1953- 1954, page 49, or Selzer and Wright, Antibiotics andChemotherapy, 6 292 (1956). Their presence may be further demonstratedby use of CH C I-l S- labeled DL-ethionine.

. The 4N demethyl 4N ethyl tetracyclines of this invention areantibacterial agents useful to combat infections due to gram-positiveand gram-negative cocci and bacilli such as pneumococci, streptococci,staphylococci and the like. The may be administered orally orparenterally in conventional dosage forms, with the dosage adjusted forthe individual needs of the one treated, in a manner similar totetracycline.

The following examples are, illustrative of the invention. Alltemperatures are expressed in degrees centigrade.

Example 1 A. Preparation of the inoculum.A sterile lyophilized pellet ofS. aureofaciens ATCC 13900 is transferred to a Distilled water to make 1liter.

and is cultured on a rotary shaker (280 r.p.m.; 2" stroke) at 25 C. forabout 72 hours.

' B. Fermentation.-To a tube containing ml. of the above medium is added1 ml. of DL-ethionine from a sterile solution containing 1 mg./ml. and 1m1. of cyanocobalamin from a sterile solution containing 400 'y/ml. Oneml. of the Streptomyces anreofaciens ATCC 13900 inoculum, prepared inPart A above, is then added to the tube and the tube is then placed on arotary shaker (280 rpm; 2" stroke) at about 25 C. for about 168 hours.C. Isolation and identification-The fermentation broth Example 2Following the procedure set forth in Example 1 but substituting in themediums of Parts A and B and equivalent amount of sodium bromide for thesodium chloride, there is produced 4N-demethyl 4N ethylbromtetracycline.

Example 3 Following the procedure set forth in Example 1 butsubstituting in Part B 1 ml. of DIS-methionine from a sterile solutioncontaining 10 mg./ml. for the cyanocobalamin there is produced 4Ndemethyl 4N ethylchlortetracycline.

Example 4 Following the procedure of Example 3 but substituting 1 ml. ofDL-methionine sulfoxide from a sterile solution containing 10 mg./ml.for the DL-methionine, there is produced4N-demethyl-4N-ethylchlortetracycline.

Example 5 Following the procedure of Example 3 but substituting 1 ml. ofDL-threonine from a sterile solution containing 10 mg./ ml. for theDL-methionine there is produced 4N- demethyl 4N ethylchlortetracycline.

Example 6 A. ,In0culum.-A sterile lyophilized pellet of Streptomycesaureofaciens ATCC 13900 is transferred to a 250 ml. Erlenmeyer flaskcontaining 50 ml. of sterile medium of the following composition:

Gm./liter Soybean meal (extraction process) 30 Glucose 50 C3003 V 7Deionized water to make 1 liter.

and is cultured on a rotary shaker (280 rpm; '2" stroke) at 25 C. forabout 72 hours.

B. Fermentati0n.-To a tube containing 10 ml. of the above medium isadded 1 ml. of DL-ethionine from a sterile solution containing -1 mgJml.and 1 ml. of cyanocobalamin from a sterile solution containing 400uy/ml.One ml. of the S. anreofaciens ATCC 13900 inoculum prepared in Part Aabove is then added to the tube and the tube is then placed on a rotaryshaker (280 r.p.m.; 2" stroke) at about 25 C. for about 168 hours.

The resultant fermentation product is then treated in accordance withthe procedures set forth in Example 1, Part C, to yield4N-demethyl-4N-ethyltetracycline.

Example 7 A. Preparation of the inocnlum.-A sterile lyophilized pelletof the microorganism S. anreofaciens, ATCC 12551, is transferred to a250 ml. Erlenmeyer flask con taining 50 ml. of sterile medium of thefollowing composition:

Distilled water to'make 1 liter.

and is cultured on a rotary shaker (280 r.p.m.; 2" stroke) at 25 C. forabout 72 hours.

B. Fermentation.-A sterile fermentation medium of the followingcomposition is prepared:

Corn starch 2m /liter 55 CaCO do 7 (Ni-10 80 d FeSO .7H O do 40 NH Cl do1.5 MnSO .4H O mg./liter 50 dO 5 ZnSO .7H O do 100 Cornsteep liquorgn1./liter 30 Cottonseed meal do 2 Lard oil percent v.v 2 Distilledwater to make 1 liter.

The medium is sterilized by autoclaving at 121 C. for 20 minutes.

To a tube containing ml. of the above sterile medium is added 2.5 mg. ofsterile DL-ethionine. One ml. of the S. aureofaciens ATCC 12551,inoculum prepared in Part A above is added to the tube and the tube isplaced on a rotary shaker (280 r.p.m.; 2" stroke) at 25 C. for about 168hours.

C. Isolation and identification-The contents of the tube obtained inPart B above is acidified to pH 2.0 by the addition of H SO shaken forabout for about ten minutes, centrifuged and the supernatant liquidchromatographically analyzed in the following chromatographic system:stationary phase, pH, 3.0, 0.3 M, phosphate buffer; moving phase,sec-butanol saturated with water on Whatman No. 1 paper to show thepresence of 4N-demethyl-4N-ethy1-6-demethylchlortetracycline.

Similarly, if L-ethionine or D-ethionine are substituted in theprocedures set forth in Example 7, for DL-ethionine, 4Ndemethyl-4N-ethyl-6-demethylchlortetracycline is also produced.

Example 8 A sterile fermentation medium of the following composition isprepared:

Deionized water to make 1 liter.

The medium is sterilized by autoclaving at 121 C. for 20 minutes.

To a tube containing 10 ml. of the above sterile medium is added 2.5 mg.of DL-ethionine. One ml. of the S. aureofaciens ATCC 12551 inoculurnprepared in Example 7, Part A, is added to the tube and the tube isplaced on a rotary shaker (280 r.p.m., 2" stroke) at 25 C. for about 168hours.

The resultant fermentation product is then treated in accordance withthe procedures set forth in Example 7, Part C, to show the presence of4N-demethyl-4N-ethyl- 6-demethy1tetracycline.

Example 9 Following the procedures of Example 8 but incorporating in thefermentation medium 1 gm./liter of sodium bromide, there is obtained 4Ndemethyl 4N ethyl-6- demethylbromtetracycline.

The invention may be variously otherwise embodied within the scope ofthe appended claim.

What is claimed is:

1. A process for preparing a compound selected from the group consistingof 4N demethyl 4N ethyl tetracycline and 4Ndemethyl-4N-ethyl(halo)tetracyclines which comprises culturing a7-chlorotetracycline producing microorganism of the strain S.aureofaciens in an aqueous nutrient medium containing an assimablesource of carbon and nitrogen in the presence of ethionine and in thefurther presence of a compound selected from the group consisting ofcyanocobalamin methionine, methionine sulfoxide and threonine, whereinsaid cyanocobalamin is present in from about 10 to mgs./1iter andmethionine, methionine sulfoxide and threonine are present in from about200 to 1500 mgs./liter; and removing the 4N demethyl 4Nethyltetracycline and 4N-demethyl 4N ethyl(ha1o)tetracycline from theculture media.

References Cited UNITED STATES PATENTS 3,172,822 3/1965 Neidleman l803,190,810 6/1965 Miller -80 3,298,927 1/ 1967 Neidleman 195- 80 OTHERREFERENCES Dulaney et al., Biochimica et Biophysica Acta, vol. 60(1962), pp. 447-449.

MAURICE W. GREENSTEIN, Primary Examiner. A! 19 MQN Q ami er,-

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 3 ,364,123 January 16 1968 Saul L. Neidleman It is hereby certified that errorappears in the above numbered patent requiring correction and that thesaid Letters Patent should read as corrected below.

Column 1, line 33, for "dimethylchlortetracycline" readdemethylchlortetracycline line 36, for "compound" read compounds line53, for "lyophobized" read lyophilized column 5, line 26, strike out"for about", second occurrence.

Signed and sealed this 18th day of March 1969 ZAL) test:

ward M. Fletcher, Jr. EDWARD J. BRENNER testing Officer Commissioner ofPatents

